Acylated protein aggregates and their use in suppressing interference in immunoassays

ABSTRACT

Subject matter of the invention are protein-containing interference-reducing agents for immunoassays consisting of protein aggregates that are acylated with --CO--R groups, wherein R is a branched or non-branched C1-C4 alkyl residue which can be substituted with hydroxy, carboxy, SO 3  H or PO 3  H 2  groups. These interference-reducing substances can be used together with a buffer as an interference-reducing agent or together with an immunological binding partner as a binding reagent to reduce non-specific interactions in immunoassays. Another subject matter of the invention is an immunological testing method wherein said interference-reducing substances are used.

This application is a rule 371 continuation of PCT/EP94/04264 filed Dec.21, 1994.

This application is a rule 371 continuation of PCT/EP94/04264 filed Dec.21, 1994.

The invention addresses acylated protein aggregates, their manufacture,and their use in an interference-reducing agent and in a binding agentfor immunoassays, and their use to reduce interference in immunoassaysas well as a corresponding immunological detection method.

Immunological detection methods have gained great importance over thelast years. They serve to detect the presence of drugs, hormones,proteins, and especially infectious organisms in biological samples in arapid and exact manner. In all immunological detection methods, there isa specific binding reaction between a first specific binding partner,the substance to be detected (analyte or ligand) and a second specificbinding partner which specifically reacts with the ligand and binds it.Ligand and specific ligand-binding partner form a specific binding pair,generally a complex between an antigen and an antibody or antibodyfragment. It is possible that more than one ligand or one bindingpartner react with each other in each reaction. These specific bindingreactions are detected in various ways. Generally, one participant inthe specific binding reaction is labelled. Conventional labellingmethods make use of radio-isotopes, chromogens, fluorogens, or enzymaticlabels. In heterogeneous immunoassays, one of the binding partners isimmobilized on a solid phase.

A difficult problem in immunoassays is that there may be undesiredinteractions and non-specific binding reactions between specific bindingpartners of the immunoassays and the sample, the additional componentscontained in the sample, and possibly the solid phase. Theseinteractions generally lead to an increase in the background signal anda higher scattering of the signals which in turn reduces sensitivity andspecificity of the test in question. Non-specific interactions with thelabelled binding partner can also lead to false-positive results whichmeans the erroneous presence of an analyte is recorded even when such ananalyte is absent.

Various attempts have been made to reduce these non-specificinteractions in immunoassays. It has been known for some time thatvarious carbohydrate components and various proteins, protein mixtures,or protein fractions as well as their hydrolysates reduce non-specificinteractions between the test components and the analytes inimmunoassays (e.g. Robertson et al., Journal of Immun. Meth. 26, 1985,EP-A-260903, U.S. Pat. No. 4,931,385). The use of protein raw fractionsand raw hydrolysates has the disadvantage that the contaminationscontained therein may also lead to interferences in the test. Moreover,enzymatically produced hydrolysates could also be contaminated withproteases used for their manufacture. Also, their quality is usually notuniform as the cleavage procedures are very difficult to control.Protease contaminations can attack test components and even minuteamounts may negatively affect the performance of the test and itsstability.

EP-A-0 331 068 describes the use of polymerized immunoglobulins (IgG) toreduce specific interfering factors, e.g. rheumatoid factors. However,non-specific interactions, especially those between labelled bindingpartners and analyte or solid phase, cannot be eliminated in asatisfactory manner. Further, the yield of human and animal IgG iscomplex and expensive.

In order to reduce non-specific interactions in immunoassays, the use ofchemically modified proteins, especially succinylated proteins, has alsobeen described (U.S. Pat. No. 5,051,356, EP-A 525916). However,especially in tests for high-molecular analytes, e.g. viral antigens,prior art does not ensure a satisfactory reduction of interferencesdespite the very high concentration of interference-reducing substances.

It was, hence, an object of the invention to provide newinterference-reducing substances and/or interference-reducing agentswhich improve the elimination of interferences in immunoassays ascompared to what is known from prior art. The invention intends inparticular to provide interference-reducing substances that generate alow blank value, a reduction of the signal scattering, and avoidfalse-positive results, especially when analyzing high molecularanalytes.

This object is accomplished by means of specifically acylated proteinaggregates.

Subject matter of the invention are protein aggregates asinterference-reducing substances for immunoassays that are acylated with--CO--R groups, wherein R is a branched or non-branched C1-C4 alkylresidue, which can be substituted with carboxy, hydroxy, SO₃ H or PO₃ H₂groups.

Another subject matter of the invention is a correspondinginterference-reducing agent for immunoassay that comprises aprotein-containing interference-reducing substance and a buffer,characterized in that it contains one or several of the acylated proteinaggregates in accordance with the invention.

Yet another subject matter of the invention is a specific bindingreagent for immunoassays, comprising a partner of a specific bindingpair, characterized in that it contains in addition one or several ofthe interference-reducing substances or interference-reducing agents inaccordance with the invention.

Yet another subject matter of the invention is a method for reducingnon-specific interactions in immunoassays by bringing theinterference-reducing substance or interference*reducing agent of theinvention in contact with the specific binding partners of a specificbinding pair used in an immunoassay.

A particular subject matter of the invention is a method for determiningimmunological ligands in a sample while reducing non-specificinteraction, by means of

1) bringing the sample to be assayed for the ligand in contact with

a) one or several interference-reducing substances, or one or severalinterference-reducing agents comprising an interference-reducingsubstance and a suitable buffer, and

b) one or several specific bindings partners of specific binding pairs,where at least one binding partner is labelled, forming a detectablebinding pair.

2) Measuring the presence or the amount of labelled binding pairs orfree labelled binding partners of a specific binding pair as a measurefor the presence or the amount of ligand in the sample, characterized inthat the interference-reducing substance is a protein aggregate acylatedwith CO-R groups, wherein R is a branched or unbranched C1-C4 alkylresidue that can be substituted with carboxy, hydroxy, SO₃ H or PO₃ H₂groups.

A ligand is a chemical or biological substance which specifically reactswith one or several corresponding specific binding partners to form acomplex. Examples include proteins, peptides, carbohydrates, toxins,haptens, drugs, viruses, fungi, and bacteria, antibodies or componentsthereof. The invention is particularly suitable for analyzing highmolecular ligands, such as viruses, virus markers, but also hormones,especially polyvalent proteins, such as HIV viruses, prostata-specificantigen (PSA), thyreotropin (TSH), carcino-embryogenic antigen (CEA),hepatitis B viruses (hepatitis B surface antigen, HBs), α-fetoprotein(AFP), human choriongonadotropin (HCG), lutenizing hormone (LH),follicle-stimulating hormone (FSH), prolactin, ferritin, insulin.

Samples are in general body fluids, such as blood, serum, or plasma,saliva, urine, or other body fluids.

A specific binding partner can be any biological or chemical bindingpartner, which reacts specifically with another biological substance toform a specific binding pair. They include antibodies, antibodyfragments, antigens, haptens, hormones, avidin, biotin, or derivativesthereof. In the present invention, preferred partners of a specificbinding pairs are antibodies or antibody fragments, which specificallybind with antigens.

At least one of the specific binding partners in an immunoassay islabelled. The labelling can furnish a measurable signal, either directlyor indirectly, e.g. through radioactivity, chemiluminescence,phosphorescence, fluorescence, or electrochemiluminescence, or a visiblecolor. The specific binding partner can also be indirectly detectable,e.g. as an enzymatic label, biotin, or avidin label, which participatein one or several reactions in order to generate a detectable substance.Enzymatic labels are preferred, especially peroxidase, glucose oxidase,β-galactosidase, or alkaline phosphatase. Another preferred label is onewith a chemiluminescent, especially electrochemiluminescent molecule.

The invention is characterized in that the interference-reducingsubstances are protein aggregates that are acylated with CO-R groups. Aprotein aggregate is understood to be an aggregate where identical ordifferent defined protein monomers were polymerized to form onehigh-molecular particle. Per definition, a protein aggregate is inparticular understood to be an artificial particle consisting of atleast 2, preferably 3 to 40,000, particularly preferred 30 to 600protein monomers. They are bound to each other in such a tight mannerthat they do not decompose into individual molecules in aqueoussolution. In a preferred manner, the protein aggregates are soluble inwater.

Polymerization and aggregation of proteins can be accomplished inthermal or chemical procedures.

In thermal polymerization procedures, protein monomers are combined intoaggregates by applying higher temperatures. The thermal polymerizationof proteins is described in EP-A-269 092, with albumin being used as anexample.

The chemical polymerization of protein monomers is accomplished withnon-protein-containing homo- or heterobifunctional linker molecules.These procedures for linking proteins are known to the experts and aredescribed in GB-A 1505 400, EP-A-0 122 209, or EP-A 269 092, forexample. Examples for the linking of protein monomers toheterobifunctional linkers are reactions withbis(maleinimido)-methylester, dimethylsuberimidate,disuccinimidyl-suberate, glutardialdehyde,N-succinimidyl-3-(2-pyridyldithio)propionate,N-5-azido-2-nitrobenzoylsuccinimide,N-succinimidyl(4-jodacetyl)-aminobenzoate or the combination ofmaleinimido-hexanoyl-N-hydroxysuccinimide ester (MHS) ormaleinimido-benzoyl-NHS (MBS) and N-succinimidyl-3-acetyl-thiopropionate(SATP). Examples for homobifunctional linkers include diaminohexane,carbodiimide, and others.

Preferred proteins are proteins with a molecular weight of more than2,000, particularly more than 10,000. Albumin or ovalbumin areparticularly preferred, especially serum albumins, and even morepreferred is bovine serum albumin.

In a preferred manner, the method of the invention makes use of proteinpolymers that were combined to aggregates in a thermal procedure.Albumin, preferably a serum albumin, especially bovine serum albumin(thermo-RSA) that was thermally polymerized and then acylated,particularly acetylated or succinilated is particularly preferred. Anon-acylated thermo-bovine serum albumin is described in EP-A 269 092.

Advantageously, polymerization is accomplished and controlled such thatpolyprotein aggregate particles of a certain largely uniform size aregenerated. A particle size of 10-200 nm, particularly advantageousbetween 20 and 50 nm is preferred. This corresponds to a molecularweight of 240,000 Da -2.2×10⁹ Da, particularly preferred 2.2×10⁶ -35×10⁶Da. The particle size can be determined in commonly known proceduressuch as PCS (Photon Correlation Spectroscopy). If necessary, theparticle size range that is particularly suitable for the invention andbe separated from a raw polymerisate mixture by means of gel filtrationin order to obtain a particularly uniform particle size.

The protein monomers used for the polymerization can either be identicalor different. It is preferred to polymerize uniform protein monomers.Albumin monomers are preferred as protein monomers. Possible albuminmonomers are all animal or human albumins, especially serum albumins.Bovine serum albumin (BSA) is particularly suitable for the invention.

In accordance with the invention, the protein aggregates are acylatedwith CO-R groups wherein R is a branched or unbranched C1-C4-alkylresidue, which can be substituted with carboxy, hydroxy, PO₃ H₂ or SO₃H. A particularly preferred substituent is the carboxy group.

The acyl groups can be either introduced in the protein monomers or inthe protein aggregates after polymerization of the protein monomers.Acylation of proteins is accomplished in accordance with known methods,preferably with acylanhydrides or with acyl-O-succinimide. Acetylated orsuccinylated protein aggregates have proven to be particularlyadvantageous, especially albumin aggregates (R═methyl and/or CH₂ --CH₂--COOH). Acetic acid-O-succinimide is preferred for acetylation.Succinic acid anhydride is preferably used for succinylation.

In the acylation process, essentially free amino groups (e.g. lysinresidues) of the protein aggregate are acylated. The term acylatedprotein aggregates means that at least one of the present free aminogroups is acylated. The nearly complete acylation of all free aminogroups is, however, preferred.

Another subject matter of the invention is an agent for reducinginterference in immunological tests, comprising a buffer forimmunological tests and the substance in accordance with the inventionfor reducing interference. Possible buffers are all aqueous bufferswhich are conventionally used in immunoassays, including phosphate,glycine-HCl, or glycine-NaOH, acetate, carbonate, citrate or organicbuffers, such as imidazole/HCl; triethanolamine; MES═(4-morpholinoethanesulfonic acid), TRIS═(TRIS(hydroxymethyl)-aminomethane),HEPES═(4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid), MOPS(3-N-morpholino-propane-sulfonic acid) and other similar buffers. The pHvalue and the concentration of the buffer salts depend on theimmunoassay involved, e.g. and also on the enzyme used for the enzymaticlabel. Usually, the pH values range between 4 and 9. Conventional bufferconcentrations are between 1 mM and 1M.

The concentration of the substance for reducing interference on theamount of immunological test components and the interfering componentscontained therein with which the agent for reducing said interference isbrought into contact. In conventional immunoassays, the concentration ofinterference-reducing substance in the interference-reducing agentshould be so high as to have a concentration between 1 mg per ml and 50mg per ml, preferably 5 mg per ml and 20 mg per ml after contact withthe immunological test components. In individual cases, concentrationsup to 200 mg per ml may also be necessary.

In addition, the interference-reducing agent of the invention can alsocontain additional substances such as preservative agents and the like.For use in an immunoassay, the interference-reducing agents isadvantageously present in an aqueous buffer solution. Moreover, it isalso possible to use it to impregnate a porous carrier material(fleece), e.g. on a test strip and its storage in a solid form, e.g. asa lyophilisate.

Another subject matter of the invention is a specific immunologicalbinding reagent with a partner of a specific binding pair, and theinterference-reducing substance or interference-reducing agent inaccordance with the invention.

In accordance with the present invention, the binding reagent isobtained such that one or several of the specific binding partners of animmunoassay and at least one interference-reducing substance orinterference-reducing agent in accordance with the invention are mixedtogether. Optionally, additives such as preservatives or stabilizers orthe like may be added. The amount of specific binding partner depends onthe immunoassay used, the amount of partner to be bound, the type oflabel used and other factors. Generally, the concentration rangesbetween 1 and 20 μg/ml.

The amount of interference-reducing substance depends on the amount oftest components of the immunoassay and interfering components containedin the binding reagent with which the binding reagent is brought intocontact. Advantageously, the concentration of interference-reducingsubstance in the binding reagent should be high enough to have a totalconcentration between 1 and 50 mg/ml, preferably 5-20 mg/ml aftercontact with the immunological test components. It is also possible thatconcentrations up to 200 mg/ml are necessary in isolated cases to havean effective reduction of interference.

The specific binding reagent can be employed in any homogeneous orheterogeneous immunoassay where a specific binding partner is useful forthe detection of the presence or absence of a specifically bindingligand. Examples include sandwich assays, competitive immunoassays andother immunoassays known to the expert. The tests can be carried out insolutions or on solid carriers.

Generally, the immunoassay in accordance with the invention is carriedout such that a sample containing the ligand is brought into contactwith the specific binding reagent that is in solution in accordance withthe present invention. A specific binding complex then forms directly orindirectly between the ligand and the specific binding partner. However,it is also possible that the ligand itself is present as a bindingreagent in accordance with the invention which is then brought intocontact with a specific binding partner or with another binding reagentin accordance with the invention. The ligand and the binding partner candirectly form a complex. The binding partner is then specific for theligand. However, it is also possible that the binding partnerforms-indirectly via one or several specific binding molecules a complexwith the ligand, and these binding molecules then bind with the ligand.

If the method is carried out as a heterogeneous immunoassay on solidcarriers, e.g. tubes, microtiter plates or a test carrier, a specificbinding partner for the ligand can be directly immobilized on thecarrier. However, it is preferred that a specific binding partner forthe ligand binding partner be immobilized on the carrier. A preferredexample is immobilized streptavidin as a specific binding reagent forbiotinylated binding partners. The various variants of suchheterogeneous immunoassays are known to the expert.

In competitive immunoassays, the ligand and the labelled ligand analogcompete for the non-labelled ligand-binding partners, which can bind tothe solid phase via a second binding site, preferably a specific bindingsite such as biotin, as is the case in heterogeneous immunoassays. Thelabelling of free and bound ligand analogs is used as a measure for thepresence or the amount of ligand to be determined.

In sandwich immunoassays, the ligand binds with a first specific bindingsite to a labelled ligand binding partner and with the second bindingsite to an unlabelled ligand binding partner, which has another specificbinding site for the solid phase, as is the case in heterogeneousimmunoassays. A complex then forms between ligands, labelled andunlabelled binding partners. In heterogeneous tests, the complex bindsto the solid phase via the unlabelled binding partner and can beseparated from the free labelled binding partner by means of washing,for example. Free or bound labelled ligand-binding partners aredetermined as a measure for the presence or the amount of the ligand tobe determined according to known methods. When enzymatic labels areused, a color-forming enzyme substrate is added to the labelled speciesand the resulting coloration is measured.

At least one of the partners of a specific immunological binding pair(ligand, labelled ligand analog, or ligand-binding partner) is presentas a binding reagent in accordance with the invention together with theinterference-reducing substance in accordance with the invention for usein an immunoassay. Advantageously, this is generally a ligand-bindingpartner, especially a labelled ligand-binding partner.

Another subject matter of the invention is the use of aninterference-reducing substance in accordance with the invention inimmunoassays.

A particular subject matter of the invention is the use of aninterference-reducing substance in accordance with the invention toreduce non-specific interactions in immunoassays.

Experience has shown that the interference-reducing substances inaccordance with the invention significantly reduce false-positivesignals of negative samples, and, hence, the blank value signal ofpositive samples. Moreover, scattering of the blank value of positivesamples and, hence, the standard deviation of the measuring value arealso reduced. This enlarges the dynamic measurement range, and themeasurement itself becomes both more sensitive and more accurate.

Yet, another subject matter of the invention is a method for preparingthe interference-reducing substances in accordance with the invention.It is characterized in that in a first step, a protein, preferablyalbumin, especially bovine serum albumin is aggregated in a chemicalaggregation reaction with bifunctional linkers; the chemical aggregationreaction preferably being a thermal aggregation reaction. The preferredparticle size ranges between 10 and 200 nm, particularly preferredbetween 20 and 50 nm. Thermal aggregation is preferably carried out at atemperature between 50 and 100° C., more particularly between 60 and 80°C. Acylation with a --CO--R-group is then carried out in a second stepwith the aid of a suitable acylation agent. The acylation shouldpreferably be completed and can be monitored via the consumption ofacylation agent in HPLC procedure, for example.

However, it is also possible that the method be carried out inversely byacylating protein according to U.S. Pat. No. 5,051,356 followed bythermal and chemical polymerization of the acylated protein.

EXAMPLE 1

Effect of acylated thermo-BSA to reduce blank values and interference

An HBsAg (hepatitis B surface antigen) sandwich immunoassay is carriedout with an HBsAg Enzymuntest® manufactured by Boehringer Mannheim.

a) Test without acylated thermo-BSA

Incubation buffer with conjugates:

40 mmol phosphate buffer pH 7.0, anti-HBsAg biotin (monoclonal, mouse)<240 ng/ml

Peptone (hydrolysate of lactalbumin): 40 mg/ml

Anti-HBsAg-POD (monoclonal, mouse), POD (peroxidase): 0.04 U/ml

Substrate/chromogen buffer:

Phosphate/citrate buffer 100 mmol/l, pH 4.4;

H₂ O₂ :3.2 mmol/l;

2,2'azino-di[3-ethyl-benzthiazoline-sulfonic acid (6)]-diammonium salt(ABTS):

1.9 mmol/l

The test is carried out on an ES 600 instrument by Boehringer Mannheim.

100 μl sample and 500 μl incubation solution with conjugate are pouredinto a streptavidin-coated tube (Enzymuntest® by Boehringer Mannheim)and incubated (180 min at 37° C.). Labelled antibodies that are notbound to the solid phase are removed from the tube by washing with 200μl washing solution.

500 μl substrate/chromogen buffer solution are added, and the colorationis measured after 60 min with a spectrophotometer at 422 nm.

b) "Interference-reducing agent" is added to the incubation buffer indifferent experiments:

1. Acylated thermo-BSA (thermally aggregated bovine serum albumin) inaccordance with the invention (2 mg/ml, particle size 30 nm)

2. Succinylated thermo-BSA in accordance with the invention (2 mg/ml,particle size 30 nm)

3. Monomeric acylated BSA in accordance with prior art (2 mg/ml)

4. Monomeric succinylated BSA (2 mg/ml) in accordance with prior art.

Table 1 shows the measurement of different samples without (example 1a)and with different incubation buffering additives 1-4 (example 1b). Whenthe interference-reducing agents (examples 1b, 1. and 2.) are used,there is a significant reduction of the blank value of the test results,and also a reduction of the standard deviation in negative sera ascompared to other additives according to prior art.

    __________________________________________________________________________    Kit Contr.            ES Buffer                    T-BSA-Ac                         T-BSA-Succ                                BSA-Ac                                     BSA-Succ    Serum panel            w/o additive                    [2 mg/ml]                         [2 mg/ml]                                [2 mg/ml]                                     [2 mg/ml]    __________________________________________________________________________    Neg. Contr.            13      0    10     28   6    Pos. Contr.            2308    3558 3739   3650 3854    Laborstandards:    1       19      18   107    51   34    2       123     192  214    209  213    3       342     551  555    5892 574    4       683     1137 1141   1195 1135    5       850     1332 1372   1384 1394    6       1053    1859 1791   1904 1748    7       1598    n.b. 2713   n.b. 2719    8       2463    4188 4091   4196 4196    Neg. Sera    BBl/27 11-13            27      13   26     37   26    BBl/27 11-14            8       3    19     19   6    BBl/27 11-17            7       1    4      28   60    BBl/27 11-18            33      7    8      22   9    BBl/2907-38            41      16   12     34   21    BBl/2907-39            48      0    8      20   53    BBl/2907-40            11      3    10     11   12    BBl/2907-41            n.b.    4    9      19   42    BBl/2907-42            5       2    17     n.b. 6    BM-91-3 8       10   5      18   7    BM-91-4 15      5    10     20   6    BM-91-29            16      6    41     33   19    BM-91-37            10      4    5      16   7    Mean (NS):            19.0    5.6  13.4   23.1 21.7    Stardard Dev.            14.6    4.7  10.4   8.0  18.9    __________________________________________________________________________

EXAMPLE 2

Lowering the blank value in an anti-HIV-P24 test

A Sandwich immunoassay is carried out using the Enzymun-Test® Anti-HIVmanufactured by Boehringer Mannheim.

Incubation buffer with conjugate:

Phosphate buffer 40 mmol/l; pH 7.0

Bovine serum components HIV-P24 antibody biotinylated (300 ng/ml)

Polyclonal anti-HIV antibody POD-labelled (100 mU/ml)

Acylated thermo-BSA, particle size 30 nm, 2 mg/ml in column 2 of table2.

Substrate buffer:

Phosphate/citrate 50 mmol/l; pH 4.4

H₂ O₂ :1.6 mmol/l

ABTS: 0.9 mmol/l

200 μl sample are incubated with 500 μl incubation buffer for 4 hours ina streptavidin tube. The sample sera do not contain HIV-P24 antigens.Subsequently, a washing step is carried out and the solution is againincubated with 700 μl substrate buffer for 1 hour. Then the measurementis carried out at 422 nm. The results are shown in table 2.

Column 1 shows the measured false-positive value (in μg/ml) withoutadding acetylated thermo-BSA in incubation buffer; column 2 shows themeasured values with acetylated thermo-BSA in incubation buffer. Column2 shows that the substance in accordance with the invention leads to areduction of false-positive signals up to 64%.

    ______________________________________                   with add.                            w/o add.    Samples        pg/ml    pg/ml    ______________________________________    Contr. Ser.    52.25    51.87    Kassel 4       13.64    2.66    Kassel 5       12.32    3.99    Kassel 6       9.36     6.80    Kassel 10      18.73    5.47    Kassel 11      18.89    7.68    Kassel 13      12.65    2.81    Kassel 16      12.65    2.81    Kassel 18      20.87    12.56    Salzburg 27    20.17    0.74    33             9.32     2.66    44             10.53    0.00    72             12.64    0.00    92             12.79    0.00    97             9.63     0.00    130            11.73    3.55    151            28.22    12.71    154            36.16    17.59    171            13.75    4.43    188            62.77    28.52    194            8.46     0.30    200            55.92    26.45    214            19.65    6.65    220            56.05    24.24    251            24.92    14.33    252            12.83    3.40    293            21.82    11.23    300            8.81     1.03    354            31.59    9.90    465            44.43    28.52    496            8.17     0.15    512            17.00    3.99    False Pos      25       9    ______________________________________

EXAMPLE 3

Reducing the interference in a PSA (prostata-specific antigen)immunoassay with the aid of acetylated thermo-BSA as compared toreducing interference with the aid of protein hydrolysate (peptone) andspecific interference-reducing proteins in accordance with prior art.

The immunoassay is carried out using the Enzymun-Test® PSA IImanufactured by Boehringer Mannheim GmbH.

1. Incubation buffer with conjugate:

Phosphate buffer 40 mmol/l, pH 7.3

MAB<PSA>mouse-PR12-Fab-POD 70 mU/ml

MAB<PSA>mouse-PR1-IgG-biotin 1 ng/ml

Various interference-reducing proteins are added to the incubationbuffer (additives 1, 2a-c in table 3).

2. Substrate buffer:

Phosphate/citrate buffer 100 mmol/l, pH 4.4

H₂ O₂ :3.2 mmol/l

Chromogen ABTS: 1.9 mmol/l

The test is carried out according to example I with 50 μm sample, 700 mlincubation buffer with conjugate (incubation time 90 min), 200 mlwashing solution, 700 μl substrate buffer (incubation time 30 min).

                                      TABELLE 3    __________________________________________________________________________          Addition of subclass-specific IgG interference                                                 w/o    Add. 1          reducing protein                       add.                                  2b Fesconasan-          .sup.2a subst. of invention                                  specimens Ent-                                          Pepton                                              w/o                                                 w/o    Add. 2          (T-BSA-II-AC)           sterptoeinpolymer                                          2c  add                                                 add.    con. samp.          0.05 mg/ml                0.10 mg/ml                      0.25 mg/ml                            0.50 mg/ml                                  0.1 mg/ml                                          5 mg/ml                                              0  0    samples          mE    mE    mE    mE    mE      mE  mE mE    __________________________________________________________________________    Standard A          10    6     3     0     89          20 20    Standard B          50    45    43    41    96          63 64    Standard C          169   170   166   173   104     160 188                                                 194    Standard D          1441  1413  1372  1512  165     1355                                              1576                                                 1545    Standard E          3578  3522  3583  3861  257     3512                                              3953                                                 3982    PS 1  13    13    8     5     43      12  25 23    PS 2  191   191   191   200   69      192 199                                                 205    PS 3  856   848   863   926   94      821 854                                                 832    PS 4  1765  1768  1789  1923  128     1719                                              1688                                                 1686    PS 5  3178  3231  3224  3515  190     3281                                              3372                                                 3299    NS 1  36    28    19    17    81      44  67 67    NS 2  25    20    14    12    76      36  38 35    NS 3  14    14    9     7     65      16  28 31    NS 4  75    69    63    62    76      33  95 92    NS 5  90    79    61    53    121     101 131                                                 128    NS 6  20    17    14    13    83      27  31 32    NS 7  21    17    12    12    57      18  32 30    NS 8  23    21    16    11    73      18  40 41    NS 9  24    21    16    13    57      13  37 38    NS 10 40    38    32    31    60      32  60 65    __________________________________________________________________________

Explanations regarding table 3

PS1 to 5:

PSA-positive human sera (male test subjects)

NS1-10:

PSA-negative human sera (sera from women)

Additive 1 is a subclass-specific IgG-polymer as aninterference-reducing protein according to EP-A-331 062 (anti-PSAantibody polyconjugate) which binds interfering components directedagainst immunological component (antibody or antibody fragmentconjugate) that generates a signal. In this example, an effect cannot beseen (see control in right column "without additive 1").

Additive 2a is the interference-reducing substance in accordance withthe invention in different concentrations (acetylated thermo-BSApolymer, particle size 30 nm). The experiments show that interference infemale sera (NS) that were measured false-positive without the additionof additive 2a is particularly effective at concentrations of 0.1 mg/ml.The dynamic measuring range is also considerably improved by reducingthe blank value (standard A) without negatively affecting the signal(calibration curve: standard A-E).

Additive 2b is an interference-reducing protein polymer in dissolvedform that is used for solid phases (thermo-BSA). This incubation bufferadditive does not show any effect on the interfering signals neither inthe example given in the table (0.1 mg per ml) nor in the higherconcentration range of 0.2 to 1.6 mg/ml. An increasing analyteconcentration (standard A-E) results in a flattening out of thecalibration curve.

Additive 2c is a protein hydrolysate in accordance with prior art(lactalbumin hydrolysate). A noticeable reduction of the interferencerequires high concentration (5 mg/ml).

EXAMPLE 4

The example is carried out corresponding to example 3; however, theincubation buffer does not contain any additive (control 1 in table 4)or succinylated thermo-BSA of various particle sizes ranging between 8nm and 74 nm at a concentration of 0.35 mg/ml.

    __________________________________________________________________________    Tab. 4    Absorcance              T-BSA-Succ                    T-BSA-Succ                          T-BSA-Succ                                T-BSA-Succ                                      T-BSA-Succ    in mA Control              8 nm .O slashed.                    18 nm .O slashed.                          30 nm .O slashed.                                36 nm .O slashed.                                      74 nm .O slashed.    __________________________________________________________________________    Standard a          41  27    15    9     14    10    Standard b          86  73    60    52    53    43    Standard c          228 215   196   183   187   164    Standard d          1602              1661  1586  1560  1535  1367    Standard e          3610              3712  3616  3657  3506  3090    St. e/a          88  137   241   408   250   309    FS  8735          94  65    34    28    26    21    FS  9623          75  51    26    23    22    18    FS  9935          73  54    23    19    17    13    FS 10070          163 149   131   105   87    68    FS 10292          294 328   262   109   76    58    FS 10310          85  56    28    25    26    23    FS 10797          77  51    23    18    18    17    FS 11009          114 91    63    53    51    49    FS 11021          99  76    44    30    28    26    FS 11033          92  68    40    33    30    28    FS  720          64  37    16    15    15    14    FS  2249          63  39    17    13    15    13    FS  3006          108 71    44    37    33    28    FS  4740          81  53    29    26    26    22    FS  8502          79  53    27    25    23    19    FS  9076          70  51    34    31    27    29    FS  9196          89  77    32    26    23    22    FS  9570          82  62    43    40    39    33    FS 10942          69  46    18    15    17    14    FS 11008          63  42    18    14    15    12    Mean  96.7              76.0  47.6  34.2  30.7  26.55    __________________________________________________________________________

EXAMPLE 5

Preparation of acetylated thermally aggregated bovine serum albumin(acetylated thermo-BSA).

1. Preparation of thermo-aggregated BSA

1 g BSA is heated up to 70° C. in 100 ml of 50 mM potassium phosphatebuffer solution (pH 7.0) and kept at this temperature for 4 hours. Thesolution is then cooled down, filtered, and concentrated to 50 mg/mlwith the aid of an ultracentrifuge (exclusion limit: 30,000 Da). Thendialysis is carried out against the 30-fold volume of redistilled waterfollowed by lyophilization. The resulting product has a molecular weightof approximately 700,000 and a particle size of 30±8 nm (measured viaphoton correlation spectroscopy (PCS)).

2. Acetylation of thermo-BSA

4000 g of thermally aggregated BSA in 100 mM potassium phosphate bufferpH 8.0 are heated up to 25° C. The protein concentration is determinedvia OD 280 nm and then the protein concentration of 10 mg/ml isadjusted. If necessary, 10 mM potassium phosphate buffer pH 8.0 can beused to adjust to the correct value. The thermo-BSA solution is pouredinto a stirring vessel and heated up to 25° C. Acetic acid inhydroxysuccinimide ester at a concentration of 100 mg/ml is dissolved inwater-free DMSO at room temperature. 11.5 ml succinimide ester solutionare added per liter of acetylated thermo-BSA solution. The so-obtainedfinal concentration in DMSO amounts to approximately 1%. After checkingthe pH value (target 6.5-9), the acetylation mixture is stirred at 25°C. for 120 min. The decrease in acetic acid N-hydroxysuccinimide esteris monitored via TSK 3000/HPLC (detection at 260 nm). After incubation,the acetylation process is stopped by adding lysine hydrochloridesolution to a final concentration of 5 mM.

The stopped acetylation reaction is filtered via a filter press. Thepress is subsequently washed with water. Filtrate and washings are thencombined.

The combined filtrate is concentrated via a polysulfone membrane 10 KDto 50 I. Concentrated solution is diafiltrated against the 10-foldvolume 20 mM potassium phosphate solution pH 7.0. The concentrate isthen diluted to twice the volume using diafiltration buffer and thenagain concentrated to the initial volume. The result of thediafiltration is determined via TSK 3000 HPLC analysis. The solution isconcentrated to 80±10 mg/ml and subsequently stabilized with 0.1%chloracetamide and 0.01% MIT (methylisothiazolone).

The PCS measurement gave a particle size of 30 nm±15.

EXAMPLE 6

Preparation of chemically polymerized, acetylated bovine serum albumin(P-BSA-Succ).

1. Polymerizing bovine serum albumin (BSA)

a) Activation of BSA with maleinimidohexanoyl-N-hydroxysuccinimide (MHS)

3 g BSA are dissolved in 30 ml of 30 mM potassium phosphate buffer, pH7.1, and 0.6 ml of a solution of 180 mg MHS/ml dimethylsulfoxide (DMSO)are added. After 1 hour incubation at 25° C., the solution is spiked upto 10 mM lysine and dialyzed against the 150-fold volume dialysis buffer(15 mM potassium phosphate buffer/50 mM NaCl/1 mM ethylenediaminetetraacetate (EDTA)/pH 6.2).

b) Activation of BSA with S-acetylthiopropionyl-N-hydroxysuccinimide(SATP)

3 g BSA are dissolved in 30 ml of 30 mM potassium phosphate buffer and0.6 ml of a solution of 140 mg SATP/ml DMSO are added. After 1 hourincubation at 25° C., the solution is spiked up to 10 mM lysine anddialyzed against the 150-fold volume of dialysis buffer (15 mM potassiumphosphate buffer/50 mM NaCl/1 mM EDTA/pH 6.2).

c) Polymerizing the activated BSA components

The solution with the SATP-activated BSA from (b) is spiked up to 25 mMhydroxylamine, a pH of 7.5 is adjusted, and incubation is carried outfor 1 hour at 25° C. Subsequently, the solution with the MHS-activatedBSA from (a) is added, and incubation is continued for another 45minutes at 25° C. Polymerization is stopped by adding 10 mM cysteine.After another 30 minutes, the solution is spiked up to 25 mMN-methylmaleinimide and dialysed against the 150-fold volume of 50 mMpotassium phosphate buffer/0.15M NaCl/pH 7.2.

2. Succinylation

2.6 ml of the solution of 0.1 g succinic acid anhydride/ml DMSO areadded to the dialyzed poly-BSA solution from (1c). After incubation for60 mm at 25° C., the solution is spiked up to 50 mM lysine, dialyzedagainst the 150-fold volume of 20 mM potassium phosphate buffer, pH 6.8,and lyophilized.

EXAMPLE 7

Reducing the interference in a Troponin-T Sandwich immunoassay withpolymerized succinylated BSA (P-BSA-Succ).

The test is carried out with the Enzymuntest® Troponin T manufactured byBoehringer Mannheim GmbH.

1. Incubation buffer with conjugate:

40 mM phosphate buffer pH 7.0

MAB<Troponin-T>M-7-Fab-POD 150 mU/ml

MAB<Troponin-T>M-11-7-IGG-biotin 2.5 μg/ml

0.5 mg/ml P-BSA-Succ; in a comparison test, the incubation buffer doesnot contain P-BSA-Succ.

2. Substrate buffer

Phosphate/citrate buffer 100 mM, pH 4.4

H₂ O₂ :3.2 mM

Chromogen ABTS 1.9 mM

The test was carried out according to example 1 with 140 μl sample and700 μl incubation buffer with conjugate (30 min incubation); 200 μlwashing solution, 700 μl substrate buffer (incubation time 15 min).

Without the addition of P-BSA-Succ to the incubation buffer, theinterfering sera listed in Table A are significantly above the cut-offvalue of 0.2 ng/ml Troponin T. The addition of 0.5 mg/ml P-BSA-Succreduces non-specific signals of all sera below the cut-off and/oreliminates some of the interference completely. The addition of theinterference-reducing protein has no significant effects on the slope ofthe calibration curve (table A). The recovery of positive human sera istherefore not affected.

Monomeric succinylated BSA (BSA-Succ) from prior art must be used in a50-fold higher concentration to obtain approximately the same reductionof interference as for polymeric, succinylated BSA (P-BSA-Succ) (tableB). The consequence thereof is the strong flattening out of thecalibration curve which in turn leads to highly elevated recoveries ofpositive human sera (table B). Monomeric BSA-Succ can therefore not beused in the Troponin-T test.

                  TABLE A    ______________________________________    Reducing the interference in a Troponin-T immunoassay with P-BSA-Succ    Cut-off: 0.2 ng/ml                                  With                Without           P-BSA-Succ    Interfering P-BSA-Succ        (0.5 mg/ml)    serum       mE     ng/ml      mE   ng/ml    ______________________________________    H 93234     90     0.392      44   0.196    R 47015     98     0.432      27   0.063    U 05706     106    0.474      21   0.003    E 02179     95     0.414      28   0.071    N 26670     90     0.39       20   <0    X 91467     104    0.462      37   0.138    R 47004     71     0.289      19   <0    X 91325     129    0.596      10   <0    G 2943      90     0.388      39   0.154    ______________________________________    Calibration curves                    Without P-BSA-Succ                                  With P-BSA-Succ    Standard        mE            mE    ______________________________________    A     0         21            13    B     0.25      63            54    C     0.77      167           145    D     4.41      857           815    E     9.84      1921          1853    F     15.4      2865          2769    ______________________________________

                  TABLE B    ______________________________________    Reducing the interference in a Troponin-T immunoassay with    monomeric BSA-Succ    Cut-off: 0.2 ng/ml            rerefence w/o                       +10 mg/ml +25 mg/ml                                         +50 mg/ml    interfering            succ       BSA succ. BSA succ.                                         BSA succ    sera    ng/ml      ng/ml     ng/ml   ng/ml    ______________________________________    11033   0.277      0.088     0.000   0.048    10292   0.443      0.195     0.147   0.129     4763   0.223      0.000     0.000   0.000    10912   0.343      0.145     0.000   0.104    ______________________________________            w/o BSA    +25 mg/ml BSA succ.                                     % deviation    ______________________________________    calib. curve            mE         mE    Standard a            16         4             25%    Standard b            44         25            57%    Standard c            162        98            60%    Standard d            956        556           58%    Standard e            1954       1168          60%    Standard f            3279       2044          82%    positive    human sera            ng/ml      ng/ml    Panel 1 1.031      1.334         129%    Panel 2 1.749      2.599         149%    Panel 3 3.915      6.223         159%    Panel 4 6.000      9.950         166%    Panel 5 9.047      14.571        161%    ______________________________________

We claim:
 1. A substance for reducing non-specific interactions inimmunoassays, comprising a protein aggregate which is acylated with--CO--R groups, wherein R is a branched or unbranched C₁₋₄ alkyl residuewhich is unsubstituted or substituted with a member selected from thegroup consisting of carboxy, hydroxy, SO₃ H or PO₃ H₂ groups.
 2. Thesubstance according to claim 1, wherein the albumin is bovine serumalbumin.
 3. The substance according to claim 1, wherein R is a methylgroup or a --CH₂ --CH₂ --COOH group.
 4. The substance according to claim1, wherein the protein aggregate is chemically aggregated with homo- orheterobifunctional linkers.
 5. The substance according to claim 1,wherein the protein aggregate is thermally aggregated.
 6. The substanceaccording to claim 5, wherein the substance is thermally aggregatedacetylated or succinylated bovine serum albumin.
 7. The substanceaccording to claim 1, wherein the particle size of the acylated proteinaggregate ranges between 10 and 200 nm.
 8. The substance according toclaim 7, wherein the particle size ranges between 20-50 nm.
 9. Aninterference-reducing reagent for reducing non-specific interactions inimmunoassays, comprising a buffer and a protein aggregate which isacylated with --CO--R groups, wherein R is a branched or unbranched C₁₋₄alkyl residue which is unsubstituted or substituted with a memberselected from the group consisting of carboxy, hydroxy, SO₃ H or PO₃ H₂groups wherein said protein aggregate is an aggregate of albumin. 10.The interference-reducing reagent according to claim 9, wherein thebuffer has a pH value between 4 and
 9. 11. A specific binding reagentfor immunoassays comprising a binding partner of a specific binding pairand a protein aggregate which is acylated with --CO--R groups, wherein Ris a branched or unbranched C₁₋₄ alkyl residue which is unsubstituted orsubstituted with a member selected from the group consisting of carboxy,hydroxy, SO₃ H or PO₃ H₂ groups wherein said protein aggregate is anaggregate of albumin.
 12. The specific binding reagent according toclaim 11, wherein the binding partner is a labeled or biotinylatedbinding partner.
 13. The specific binding reagent according to claim 11,wherein the binding partner is an antigen, antibody, or antibodyfragment.
 14. The specific binding reagent according to claim 13,wherein the binding partner is an antibody or antibody fragment with anenzymatic or electrochemiluminescent label.
 15. A method for determiningthe presence of an immunological ligand in a sample, wherein the methodreduces non-specific interactions, comprising the steps ofcontacting asample to be tested for a ligand with (a) at least one protein aggregatewhich is acylated with --CO--R groups, wherein R is a branched orunbranched C₁₋₄ alkyl residue which is unsubstituted or substituted witha member selected from the group consisting of carboxy, hydroxy, SO₃ Hor PO₃ H₂ groups wherein said protein aggregate is an aggregate ofalbumin; and (b) at least one specific binding partner of a specificbinding pair, wherein the at least one binding partner is labeled, toform a detectable binding pair, and determining the presence or amountof the labeled binding pair or the free labeled binding partner of aspecific binding pair as a measure of the presence or concentration ofthe ligand in the sample.
 16. The method according to claim 15, whereinthe ligand is a high molecular weight ligand.
 17. The method accordingto claim 16, wherein the ligand is selected from the group consisting ofa virus, virus marker, tumor marker, and a hormone.
 18. The methodaccording to claim 15, wherein said binding partner of a specificbinding pair is selected from the group consisting of antigens,antibodies, and antibody fragments.
 19. The method according to claim15, wherein the at least one binding partner carries an enzymatic orelectrochemiluminescent label.
 20. The method according to claim 15,wherein the at least one specific binding partner has a second specificbinding site for a solid phase-bound binding partner.
 21. The methodaccording to claim 20, wherein the at least one binding partner isbiotinylated and capable of binding to a streptavidin surface.
 22. Amethod for reducing nonspecific interference in an immunoassay whichuses at least one buffer, comprising adding a protein aggregate which isacylated with --CO--R groups, wherein R is a branched or unbranched C₁₋₄alkyl residue which is unsubstituted or substituted with a memberselected from the group consisting of carboxy, hydroxy, SO₃ H or PO₃ H₂groups wherein said protein aggregate is an aggregate of albumin, to abuffer used in said immunoassay.
 23. A method for preparing aninterference-reducing substance, comprising the steps of a) polymerizinga protein to form an aggregate, and b) acylating the protein aggregatewith ah acylating agent wherein the protein is albumin.
 24. The methodaccording to claim 23, wherein the protein is thermally polymerized. 25.The method according to claim 23, wherein the protein aggregate isacetylated with acetyl-o-succinimide or with succinic acid anhydride.26. A kit for decreasing nonspecific interactions in an immunoassay,comprising (a) a protein aggregate which is acylated with --CO--Rgroups, wherein R is a branched or unbranched C₁₋₄ alkyl residue whichis unsubstituted or substituted with a member selected from the groupconsisting of carboxy, hydroxy, SO₃ H and PO₃ H₂, and (b) a bufferwherein said protein aggregate is an aggregate of albumin.
 27. The kitaccording to claim 26, further comprising at least one binding partnerspecific for a high molecular weight ligand to be detected in saidimmunoassay.
 28. The kit according to claim 27, wherein said bindingpartner is detectably labeled.
 29. The kit according to claim 28,further comprising at least one unlabeled binding partner specific for ahigh molecular weight ligand to be detected.
 30. A test strip fordetecting a ligand, comprising a specific binding partner and a proteinaggregate which is acylated with --CO--R groups, wherein R is a branchedor unbranched C₁₋₄ alkyl residue which is unsubstituted or substitutedwith a member selected from the group consisting of carboxy, hydroxy,SO₃ H and PO₃ H₂ ; wherein said specific binding partner, said proteinaggregate or both said specific binding partner and said proteinaggregate are reversibly impregnated on different layers wherein saidprotein aggregate is an aggregate of albumin.
 31. A test strip fordetecting a ligand, comprising a specific binding partner and a proteinaggregate which is acylated with --CO--R groups, wherein R is a branchedor unbranched C₁₋₄ alkyl residue which is unsubstituted or substitutedwith - a member selected from the group consisting of carboxy, hydroxy,SO₃ H and PO₃ H₂ ; wherein said specific binding partner and proteinaggregate are, reversibly impregnated on the same layer which is fixedon a carrier wherein said protein aggregate is an aggregate of albumin.